JP2005290610A - Nanoscale fiber and formed product of polysaccharides - Google Patents
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本発明は、静電紡糸法によって得る多糖類のナノスケールの繊維、その薄膜等の成形体に関する。より詳細には本発明は、医療分野の医用材料、特に、再生医療分野における生体組織培養の足場として用いることができ、また、生体材料（人工弁、人工臓器、人工血管、創傷被覆材等）の一部として生体組織の皮膚や臓器等の修復、再生、治療に用いることができる。また、多糖類のナノスケールの繊維によって構成された不織性の3次元構造を有する薄膜に関する。 The present invention relates to a molded article such as a nanoscale fiber of a polysaccharide obtained by an electrospinning method and a thin film thereof. More specifically, the present invention can be used as a medical material in the medical field, in particular, as a scaffold for culturing living tissue in the field of regenerative medicine, and a biomaterial (artificial valve, artificial organ, artificial blood vessel, wound dressing, etc.) As a part of this, it can be used for repairing, regenerating and treating skin and organs of living tissues. The present invention also relates to a thin film having a non-woven three-dimensional structure constituted by polysaccharide nanoscale fibers.
再生医療は、1998年の米国において、骨、軟骨、皮膚、神経、筋肉、血管や各臓器への再生能力の可能性を秘めたヒト胚性幹（ES）細胞が樹立され、ゲノム医療と並んで注目されている分野である。再生医療は、細胞や組織を培養し、機能を失った生体組織を再生させたり、臓器機能を代替したりする生体組織工学的な技術分野で新しい医療である。再生医療の生体組織工学で重要なアプローチが再生の場の構築である。再生の場には、水溶性高分子素材や生体材料が多く使われている。機能性に優れた足場材料の開発がこれまで以上に求められている。
一般に、医用材料として生体材料に要求される条件は、生体機能性（用途に応じた性能や効果）、生体安全性（毒性がない）、可滅菌性（感染防止のための滅菌ができる）、生体適合性（生体に対する親和性）がある。また、生体組織培養および生体材料の一部として、細胞や組織の足場となる条件には、上記の条件以外に、１）適度な力学的強度、２）生体吸収性、３）細胞接着における十分な比表面積の確保、４）栄養や酸素供給が可能な隙間構造、等が挙げられる。
Regenerative medicine was established in 1998 in the United States, where human embryonic stem (ES) cells with the potential to regenerate bone, cartilage, skin, nerves, muscles, blood vessels and other organs were established. This is a field that is attracting attention. Regenerative medicine is a new medical technique in the field of biological tissue engineering that cultivates cells and tissues, regenerates biological tissue that has lost its function, or substitutes for organ function. An important approach in tissue engineering for regenerative medicine is the establishment of a regenerative field. Many water-soluble polymer materials and biomaterials are used for regeneration. The development of scaffold materials with excellent functionality is required more than ever.
In general, the requirements for biomaterials as medical materials are biofunctionality (performance and effects according to the application), biosafety (non-toxic), sterilizable (can be sterilized to prevent infection), There is biocompatibility (affinity to the living body). In addition to the above conditions, as a part of biological tissue culture and biomaterials, there are 1) adequate mechanical strength, 2) bioabsorbability, and 3) sufficient cell adhesion. Securing a specific surface area, and 4) a gap structure capable of supplying nutrients and oxygen.
生体内での細胞組織の足場は、コラーゲンやムコ多糖類（生体内ではタンパク質と結合しプロテオグリカンとして存在）に代表される細胞外物質（細胞外マトリクス）であり、細胞が増殖し正常機能を発揮するために必要な細胞接着に深く関っている。
天然高分子として多糖類は、生体親和性が高く、細胞毒性がないばかりでなく、成形加工性もあって、医用材料の素材として注目されている。この多糖類を素材として、適度な力学的強度があって、細胞との親和性が高く、細胞接着における比表面積を十分に確保でき、栄養や酸素供給可能な密度を有する材料が得ることができれば、従来からあるコラーゲンや合成高分子素材からなる材料の代替として利用できるばかりでなく、これまで以上に細胞との界面制御の可能性が広がる。
Cellular tissue scaffolds in vivo are extracellular substances (extracellular matrix) typified by collagen and mucopolysaccharides (which bind to proteins and exist as proteoglycans in vivo), and cells proliferate and perform normal functions. It is deeply involved in cell adhesion necessary to do.
As a natural polymer, polysaccharides are attracting attention as raw materials for medical materials because they are not only highly biocompatible and non-cytotoxic, but also have moldability. If this polysaccharide can be used as a raw material, a material having a suitable mechanical strength, a high affinity with cells, a sufficient specific surface area for cell adhesion, and a density capable of supplying nutrients and oxygen can be obtained. Not only can it be used as a substitute for conventional collagen and synthetic polymer materials, but also the possibility of interfacial control with cells is expanded more than ever.
細胞と界面制御する手段としてナノテクノロジーが用いられはじめている。ナノテクノロジーとは、1nm〜100nmの規模の原子や分子を操作・制御して、物質の構造や配列を変え、新しい機能やより優れた特性を作り出すことができる革新的技術として注目されており、その発展は目覚しいものがあって、ITやエレクトロニクス、バイオなど様々な分野で着々と実用化が進められている（非特許文献１および２）。繊維の世界では、従来の実用繊維が20〜50μmとすると、最近では合成高分子素材で海島型等の複合紡糸技術による極細繊維、超極細繊維と呼ばれる、平均直径が2〜5μm、いわゆるマイクロファイバーの製品化やより細い繊維（サブミクロンサイズ以下）の実用化に向けた開発が進められている段階である。一般に平均直径が100nm以下（1mmの1万分の１以下）、いわゆるナノサイズになると、比表面積が各段に大きくなることが知られている（非特許文献３）。しかし、現状の技術では天然素材の分野において、工業規模でナノスケール〜サブミクロンスケールの繊維を紡糸できる技術は未だ確立されていない。 Nanotechnology is beginning to be used as a means to control the interface with cells. Nanotechnology is attracting attention as an innovative technology that can manipulate and control atoms and molecules on the scale of 1 nm to 100 nm, change the structure and arrangement of materials, and create new functions and superior properties. Its development has been remarkable, and its practical application is steadily being promoted in various fields such as IT, electronics, and biotechnology (Non-patent Documents 1 and 2). In the fiber world, if the conventional practical fiber is 20-50 μm, it is a so-called microfiber with an average diameter of 2-5 μm, which is recently called a synthetic polymer material called ultra-fine fiber or ultra-fine fiber by a composite spinning technique such as sea-island type. Development is underway for commercialization of products and practical application of finer fibers (submicron size or less). In general, when the average diameter is 100 nm or less (less than 1 / 10,000 of 1 mm), so-called nano-size, it is known that the specific surface area increases in each step (Non-patent Document 3). However, with the current technology, in the field of natural materials, a technology capable of spinning nanoscale to submicron scale fibers on an industrial scale has not yet been established.
最近、静電紡糸の技術を用いた方法がナノスケールの繊維を得るための手段として注目されている。静電紡糸法は今から70年以上も昔に考案され、繊維やフィルム、不織布をつくる技術として開発されたものであるが、現在、質量分析でも応用されている技術である。特にこの方法によって紡糸する製造方法をエレクトロスプレースピニング（ESP）法とも呼び、また得られる薄膜や不織布の製造法をエレクトロスプレーデポジション（ESD）法とも呼んでいる（非特許文献４）。
その特徴は、従来の商業的紡糸方法とは異なり、高電圧下で高分子素材を帯電させ表面反発力によって紡糸する方法である。これまでに静電紡糸法で応用検討された素材としては、合成高分子系ではポリビニルアルコール、ポリエチレンオキシド、ポリビニルピロリドン、ポリアクリルアミド、ポリウレタン、ポリカーボネート、ポリテトラフルオロエチレン、ポリエチレン、ポリプロピレン、ポリアクリレート、ポリヒドロキシブチレート、ポリアニリン、アラミド系等があり、天然高分子系ではデオキシリボ核酸、コラーゲン、α-ラクトアルブミン、インベルターゼ、絹フィブロイン等がある。しかし、多くの形成される繊維は、素材によって溶液の物性パラメータとして濃度や分子量、粘度、表面張力、電気伝導度、誘電率、印加電圧、湿度、等が異なっており、多糖類の条件を予測することは極めて困難であった。
Recently, a method using an electrospinning technique has attracted attention as a means for obtaining nanoscale fibers. The electrospinning method was devised more than 70 years ago, and was developed as a technique for producing fibers, films, and non-woven fabrics. Currently, it is also applied to mass spectrometry. In particular, a production method for spinning by this method is also called an electrospray spinning (ESP) method, and a method for producing a thin film or nonwoven fabric obtained is also called an electrospray deposition (ESD) method (Non-patent Document 4).
Its characteristic is that, unlike the conventional commercial spinning method, the polymer material is charged under high voltage and spun by surface repulsion. The materials that have been studied for application in the electrospinning method so far include polyvinyl alcohol, polyethylene oxide, polyvinyl pyrrolidone, polyacrylamide, polyurethane, polycarbonate, polytetrafluoroethylene, polyethylene, polypropylene, polyacrylate, polyacrylate for synthetic polymer systems. There are hydroxybutyrate, polyaniline, aramid and the like, and natural polymers include deoxyribonucleic acid, collagen, α-lactalbumin, invertase, silk fibroin and the like. However, many formed fibers have different concentrations, molecular weights, viscosities, surface tensions, electrical conductivities, dielectric constants, applied voltages, humidity, etc. as physical properties parameters of solutions depending on the materials, and predict the conditions of polysaccharides. It was extremely difficult to do.
キチンやキトサン、ヒアルロン酸、コンドロイチン硫酸等の多糖類は細胞外物質としてコラーゲンと同様に細胞の増殖や分化の足場の素材として重要であり、医用材料として利用できる可能性が考えられる。天然高分子で優れた機能を有する多糖類の静電紡糸について若干の言及はあるが実施例で裏付けされたものではなく（特表2003-521493号公報）、また、天然有機物の超微細化繊維について、繊維を溶媒中に分散しせん断力によって得るもので繊維径が均一なナノスケールの繊維を得ることはできず（特開2003-155349号公報）、ナノスケールの粒子のキトサンに関して記述はあるが製法は静電紡糸ではない（特表2002-536392号公報）。以上のように製法や性状が異なる種々のナノスケールの物体はあるが、静電紡糸法によってできた多糖類のナノスケールの繊維や薄膜についてはこれまでのところなかった。静電紡糸法によれば、ナノスケールの粒子や繊維などが作製できるばかりでなく、これらが集積されて多孔性や不織性の薄膜もできる。また、表面に様々なコーティング加工もでき応用展開が可能である。 Polysaccharides such as chitin, chitosan, hyaluronic acid, and chondroitin sulfate are important as an extracellular substance as a scaffold for cell growth and differentiation, like collagen, and may be used as medical materials. Although there are some references to the electrospinning of polysaccharides that have excellent functions in natural polymers, they are not supported by the examples (Japanese Patent Publication No. 2003-521493), and natural organic ultrafine fibers In regard to the above, it is possible to obtain nanoscale fibers having a uniform fiber diameter obtained by dispersing fibers in a solvent and shearing force (Japanese Patent Laid-Open No. 2003-155349), and there is a description of nanoscale particle chitosan However, the production method is not electrostatic spinning (Japanese Patent Publication No. 2002-536392). As described above, there are various nanoscale objects having different production methods and properties, but there have been no nanoscale fibers and thin films of polysaccharides produced by the electrospinning method so far. According to the electrospinning method, not only nanoscale particles and fibers can be produced, but these can be integrated to form a porous or non-woven thin film. In addition, various coating processes can be applied to the surface and application development is possible.
本発明は、医療分野の医用材料、特に、再生医療における生体組織培養の基材および生体組織の欠損、修復、再生、治療を目的とした生体材料（人工弁、人工臓器、人工血管、創傷被覆材等）の一部として、細胞組織の足場や支持体であって、酸素や栄養素の供給にも優れ、効率良く細胞の増殖や分化が期待でき、素材が多糖類でナノスケールの繊維であり、さらにはそれを構成し得られる不織性の薄膜、およびそれらで加工した成形体を提供することを目的としている。 The present invention relates to medical materials in the medical field, in particular, biological materials for biological tissue culture in regenerative medicine and biological materials for the purpose of defect, repair, regeneration, and treatment of biological tissues (artificial valves, artificial organs, artificial blood vessels, wound coverings). As a part of the material, etc., it is a scaffold and support for cellular tissue, excellent in supplying oxygen and nutrients, and can be expected to grow and differentiate efficiently, and the material is polysaccharide and nanoscale fiber Furthermore, it is an object to provide a non-woven thin film that can be formed from the thin film, and a molded body processed with the non-woven thin film.
本発明者は、上記課題を解決するために、鋭意に検討した結果、動物、植物、海藻、微生物等に含まれる多糖類を素材に用いて、従来の商業的な紡糸方法ではなく、本発明者が先に出願した装置（出願番号;特願2003‐040642）を使用して、静電紡糸法によって、多糖類を素材としたナノスケールの繊維（ナノファイバー）が得られることを見出した。この技術によれば、多糖類からなるナノスケールの繊維で構成され、薄く緻密で不織性を示し、3次元構造で高い比表面積を有する薄膜が得られることを確認し、本発明に至った。すなわち、本発明は、静電紡糸法による多糖類のナノスケールの繊維および成形体を特徴とする。 As a result of diligent studies to solve the above-mentioned problems, the present inventor uses polysaccharides contained in animals, plants, seaweeds, microorganisms, etc. as materials, and does not use conventional commercial spinning methods, but the present invention. The present inventors have found that nanoscale fibers (nanofibers) made of polysaccharides can be obtained by the electrospinning method using the device (application number; Japanese Patent Application No. 2003-040642) filed earlier. According to this technology, it was confirmed that a thin film composed of nanoscale fibers made of polysaccharides, showing a thin, dense and non-woven property, and having a high specific surface area with a three-dimensional structure was obtained. . That is, the present invention is characterized by polysaccharide nanoscale fibers and molded bodies by an electrospinning method.
本発明は、以下の（１）〜（３）の多糖類のナノスケールの繊維を要旨としている。
（１）静電紡糸法によって得られる多糖類を主原料とする繊維であって、直径が500nm以下であることを特徴とする多糖類のナノスケールの繊維。
（２）多糖類以外の添加物を含む複合組成物あるいは多糖類単独の繊維である上記（１）の多糖類のナノスケールの繊維。
（３）直径が1〜100nmである上記（２）の多糖類のナノスケールの繊維。
The gist of the present invention is the nanoscale fiber of the following polysaccharides (1) to (3).
(1) A nanoscale fiber of a polysaccharide, which is a fiber mainly composed of a polysaccharide obtained by an electrospinning method and having a diameter of 500 nm or less.
(2) A nano-scale fiber of a polysaccharide according to the above (1), which is a composite composition containing additives other than polysaccharides or a single fiber of polysaccharides.
(3) The nanoscale fiber of the polysaccharide according to the above (2) having a diameter of 1 to 100 nm.
本発明は、以下の（４）〜（５）の不織性の薄膜を要旨としている。
（４）上記（１）の繊維からなる不織性の薄膜。
（５）上記（１）の繊維および直径500nmを超えるミクロ繊維の集合体からなる不織性の薄膜。
The gist of the present invention is the following non-woven thin film (4) to (5).
(4) A non-woven thin film comprising the fiber of (1) above.
(5) A non-woven thin film comprising an assembly of the fibers of (1) above and microfibers having a diameter exceeding 500 nm.
本発明は、以下の（６）〜（７）の医用材料を要旨としている。
（６）上記（１）の繊維を含む医用材料。
（７）上記の（４）または（５）の不織性の薄膜を含む医用材料。
The gist of the present invention is the following medical materials (6) to (7).
(6) A medical material containing the fiber of (1) above.
(7) A medical material comprising the non-woven thin film of (4) or (5) above.
本発明は、静電紡糸法によって得られる多糖類のナノスケールの繊維であって、安全性が高く、細胞毒性がなく、比表面積が大きく、その薄膜、および医用材料等の成形体を提供することができる。
特に、再生医療における生体組織培養の基材および生体組織の欠損、修復、再生、治療を目的とした生体材料（人工弁、人工臓器、人工血管、創傷被覆材等）の一部として用いられ、細胞組織の足場や支持体であって、酸素や栄養素の供給にも優れ、効率良く細胞の増殖や分化が期待できる。
The present invention provides polysaccharide nano-scale fibers obtained by an electrospinning method, which is highly safe, non-cytotoxic, has a large specific surface area, a thin film thereof, and a molded article such as a medical material. be able to.
In particular, it is used as a part of biological tissue culture substrate in regenerative medicine and biological materials (artificial valve, artificial organ, artificial blood vessel, wound dressing, etc.) for the purpose of defect, repair, regeneration, treatment of biological tissue, It is a scaffold and support for cellular tissues, and is excellent in supplying oxygen and nutrients, and can be expected to proliferate and differentiate cells efficiently.
以下、本発明について詳しく説明するが、本発明に関して何ら拘束されるものではない。
本発明は、再生医療における生体組織培養の基材および代替皮膚や欠損組織の修復、再生、治療を目的とした生体材料（人工弁、人工臓器、人工血管、創傷被覆材等）の一部であり、細胞組織の足場や支持体であって、酸素や栄養素の供給にも優れ、効率良く細胞の増殖や分化が期待できる。多糖類でナノスケールの繊維であり、さらには不織性の薄膜、およびそれらで加工された医用材料等の成形体に関する。
すなわち、本発明の多糖類繊維、平均直径が数nm〜数100nmのナノスケールの繊維、いわゆるナノファイバーであるが、静電紡糸法によって連続的に大規模に紡糸できるもので、例えば図１記載のような装置を用いることで達成できる。その装置には、電極（白金線等）を挿入したキャピラリー（ガラス製等）、導電性の基板（アルミニウム製等）等が装備されている。その原理は、大気圧下で、高分子溶液の入ったキャピラリーのノズル先端と基板間に、2,000V〜20,000Vの高電圧を印加すると、ノズル先端で高分子溶液の電気的反発力が表面張力よりも高くなって噴射され、条件によって粒子状や紡錘状、繊維状の構造体が乾燥されて基板上に集積される。
Hereinafter, although this invention is demonstrated in detail, it is not restrained at all regarding this invention.
The present invention is a part of a biological tissue culture base material in regenerative medicine and a biomaterial (an artificial valve, an artificial organ, an artificial blood vessel, a wound dressing material, etc.) for the purpose of repair, regeneration, and treatment of alternative skin and defective tissue. Yes, it is a scaffold or support for cellular tissue, and is excellent in supplying oxygen and nutrients, and can be expected to proliferate and differentiate cells efficiently. The present invention relates to polysaccharides and nanoscale fibers, and further relates to a non-woven thin film, and a molded article such as a medical material processed with them.
That is, the polysaccharide fiber of the present invention is a nanoscale fiber having an average diameter of several nanometers to several hundred nanometers, so-called nanofiber, which can be continuously spun on a large scale by an electrostatic spinning method. This can be achieved by using a device such as The apparatus is equipped with a capillary (such as glass) into which an electrode (platinum wire or the like) is inserted, a conductive substrate (such as aluminum), or the like. The principle is that when a high voltage of 2,000 V to 20,000 V is applied between the nozzle tip of the capillary containing the polymer solution and the substrate under atmospheric pressure, the electric repulsive force of the polymer solution at the nozzle tip causes surface tension. The particles, spindles, and fibrous structures are dried and accumulated on the substrate depending on conditions.
静電紡糸法によって形成される繊維には、高分子素材の分子量、溶液の濃度、粘度、温度、表面張力、電気伝導度、誘電率等の物性、印加電圧、キャピラリー先端口径等のパラメータの条件があって高分子素材によって異なる。本発明においても、これらの条件と用いる溶媒や添加物の組み合わせによって繊維状物が得られる最適な範囲を見出した結果、多糖類素材で、直径が数nm〜数100nmであるナノスケールの繊維とそれらで構成される2次元的、3次元的に広がりを持った不織性の薄膜が安定的に得ることができた。 For fibers formed by electrostatic spinning, the molecular weight of the polymer material, solution concentration, viscosity, temperature, surface tension, electrical conductivity, physical properties such as dielectric constant, applied voltage, capillary tip diameter and other parameter conditions Depending on the polymer material. Also in the present invention, as a result of finding an optimum range in which a fibrous material can be obtained by a combination of these conditions and the solvent and additives used, it is a polysaccharide material, and a nanoscale fiber having a diameter of several nanometers to several hundred nanometers. A non-woven thin film with a two-dimensional and three-dimensional extent composed of them could be obtained stably.
本発明に関る高分子素材とは多糖類である。多糖類とは、植物や海藻、甲殻、微生物、キノコなどに存在するもので特に限定するものではない。また、多糖類の種類は、一般に大別すると天然物と半合成品に分けられるが特に限定するものではない。多糖類はグルコース（水酸基をもつ中性糖やアミノ基やカルボキシル基などの官能基をもつアミノ糖や酸性糖等もある）等の単糖がグリコシド結合により多く連なった形のもので、一般に、細胞毒性がなく、構成糖や官能基、分子量によってそれぞれ固有の性質を持ち、なおかつ資源が豊富にある。多糖類はすべての生物に存在する重要な成分であるが、単なる細胞間の充填や組織の支持体としての役割以外にも、細胞間の情報伝達や細胞認識（自己非自己、免疫）、組織構築、発生や細胞分割等に深くかかわっている。また、その他にも抗ガン、抗ウイルス、免疫賦活、抗炎症等の機能が知られている。これら多糖類は、安全面で高く評価されているので、食品素材をはじめ、医用素材や医薬素材等に広く利用されている。 The polymer material according to the present invention is a polysaccharide. The polysaccharide is present in plants, seaweeds, shells, microorganisms, mushrooms and the like and is not particularly limited. The types of polysaccharides are generally classified into natural products and semi-synthetic products, but are not particularly limited. A polysaccharide is a form in which monosaccharides such as glucose (there are also neutral sugars having hydroxyl groups, amino sugars having functional groups such as amino groups and carboxyl groups, and acidic sugars) linked together by glycosidic bonds. It is non-cytotoxic, has unique properties depending on the constituent sugar, functional group, and molecular weight, and is rich in resources. Polysaccharides are important components that exist in all living organisms, but besides simple cell-to-cell filling and role as tissue supports, intercellular communication, cell recognition (self-nonself, immunity), tissues Deeply involved in construction, development and cell division. In addition, other functions such as anti-cancer, anti-virus, immunostimulation, and anti-inflammation are known. Since these polysaccharides are highly evaluated in terms of safety, they are widely used for food materials, medical materials, pharmaceutical materials, and the like.
例えば、静電紡糸法で用いられる素材としては、動物性ではキチン、キトサン、ヒアルロン酸、コンドロイチン硫酸、ヘパリン、ケラト硫酸等のムコ多糖がある。植物系では、セルロース、ペクチン、キシラン、リグニン、グルコマンナン、ガラクツロン、サイリウムシードガム、タマリンド種子ガム、アラビアガム、トラガントガム、大豆水溶性多糖等がある。海藻系では、アルギン酸、カラギーナン、ラミナラン、寒天(アガロース)、フコイダン等がある。カビやきのこ等を含む微生物系では、プルラン、デキストラン、カードラン、レンチナン、キサンタンガム等がある。また、それら多糖類の誘導体も利用できる。 For example, as materials used in the electrospinning method, there are mucopolysaccharides such as chitin, chitosan, hyaluronic acid, chondroitin sulfate, heparin, and keratosulfuric acid. In plant systems, there are cellulose, pectin, xylan, lignin, glucomannan, galacturon, psyllium seed gum, tamarind seed gum, gum arabic, tragacanth gum, water soluble polysaccharides of soybeans, and the like. In the seaweed system, there are alginic acid, carrageenan, laminaran, agar (agarose), fucoidan and the like. Microbial systems including molds and mushrooms include pullulan, dextran, curdlan, lentinan, xanthan gum and the like. In addition, derivatives of these polysaccharides can also be used.
これら多糖類の素材は溶解してもそのまま懸濁しても構わないが、好ましくは溶解する方がよい。その理由としては、溶液であれば、多糖類がナノ繊維の中心から表面にかけて均一に分散し、繊維の機能を最大限に発揮することができる。一方、多糖類を懸濁する場合には、ナノ繊維よりも小さくし均一な粒子が必要であり、そのような粒子をまず製造することは非常に困難で実用的とは言えない。多糖類の溶媒としては、水、酸、アルカリ、エタノール、メタノール、アセトン、トルエン等があげられるが、多糖類の溶解性を考慮して選択すればよく、少なくとも1種類、あるいは溶液の物性を変えるために複数使用しても何ら問題にはならないが、好ましくは弱酸〜中性〜弱アルカリの溶媒であって、精製水やエタノール水、生理食塩水、各種緩衝液（リン酸緩衝液、酢酸緩衝液等）等が望ましい。また、多糖類の分子量としては、多糖類の種類や抽出方法によって一定でなく、分子量が数千のものもあれば、数万から数100万のものもあって、特に限定するものではない。一般に分子量が大きいほど繊維構造を形成しやすく強度のある繊維が得やすく、分子量が小さいほど粒子構造を形成しやすく、好ましくは分子量が1万〜500万で、最も好ましくは3万〜300万である。溶液の粘度としては、分子量が大きいほど、濃度が高いほど粘度が高く、溶液の温度やpHでも変わるが、一般に粘度が高いほど細く長い繊維が得やすく、粘度が低いほど細く短い繊維や粒子が得やすく、好ましくは1,000mPa・s以下であり、最も好ましくは500mPa・s以下である。多糖類の濃度としては、粘度が1,000mPa・sを超えなければ特に限定するものではないが、一般に濃度が高いほど繊維構造を得やすく、濃度が低いほど細く短い繊維や粒子が得やすく、好ましくは0.5%〜50%であり、最も好ましくは1%〜30％である。また、溶液の電気伝導度や誘電率等の物性は、紡糸した際の荷電状態に影響するが、一般に電気伝導度が低いほど静電紡糸法に適しており、多糖類の溶液を透析膜や電気透析、UF膜やNF膜、RO膜等で脱塩処理するか、あるいは多糖類をエタノールやアセトンなどを用いて溶媒沈殿させて洗浄してから用いるのが望ましい。溶液の電気伝導度としては、好ましくは600mS/cm以下であり、最も好ましくは100mS/cm以下である。溶液の温度は、多糖類や含まれる添加物の機能や活性が損なわない限りにおいては、特に温度を制限するものではなく、室温でも冷やしても加熱しても構わないが、好ましくは0〜100℃、最も好ましくは4〜80℃である。これら多糖類の溶液は、不溶解物等がある場合には遠心分離あるいはメンブランフィルターで濾過すればよい。例えば、遠心分離であれば、3,000rpm・10分間以上、メンブランフィルターであれば、0.2μm〜10μmの孔径で処理すればよい。 These polysaccharide materials may be dissolved or suspended as they are, but it is preferable to dissolve them. The reason is that in the case of a solution, the polysaccharide is uniformly dispersed from the center to the surface of the nanofiber, and the function of the fiber can be exhibited to the maximum. On the other hand, when suspending polysaccharides, uniform particles are required that are smaller than nanofibers, and it is very difficult and impractical to produce such particles first. Examples of the polysaccharide solvent include water, acid, alkali, ethanol, methanol, acetone, toluene, and the like. The polysaccharide may be selected in consideration of the solubility of the polysaccharide, and at least one kind or the physical properties of the solution is changed. For this reason, it is not a problem even if it is used multiple times, but it is preferably a weak acid-neutral-weak alkali solvent, purified water, ethanol water, physiological saline, various buffers (phosphate buffer solution, acetate buffer) Liquid etc.) is desirable. Further, the molecular weight of the polysaccharide is not particularly limited depending on the type of polysaccharide and the extraction method, and there are several molecular weights and several tens of thousands to several millions. In general, the higher the molecular weight, the easier it is to form a fiber structure that is easy to form, and the smaller the molecular weight, the easier it is to form the particle structure, preferably the molecular weight is 10,000 to 5,000,000, most preferably 30,000 to 3,000,000. is there. As the viscosity of the solution, the higher the molecular weight, the higher the concentration, the higher the viscosity, and the temperature and pH of the solution also vary. Generally, the higher the viscosity, the easier to obtain thin and long fibers, and the lower the viscosity, the thinner and shorter fibers and particles. It is easy to obtain, preferably 1,000 mPa · s or less, and most preferably 500 mPa · s or less. The concentration of the polysaccharide is not particularly limited as long as the viscosity does not exceed 1,000 mPa · s. In general, the higher the concentration, the easier it is to obtain a fiber structure, and the lower the concentration, the easier to obtain fine and short fibers and particles. Is 0.5% to 50%, most preferably 1% to 30%. In addition, physical properties such as the electrical conductivity and dielectric constant of the solution affect the charged state at the time of spinning, but generally the lower the electrical conductivity, the more suitable for the electrospinning method. It is desirable to use after desalting with electrodialysis, UF membrane, NF membrane, RO membrane or the like, or washing the polysaccharide with ethanol or acetone for precipitation. The electric conductivity of the solution is preferably 600 mS / cm or less, and most preferably 100 mS / cm or less. The temperature of the solution is not particularly limited as long as the function and activity of the polysaccharide and the additive contained therein are not impaired, and may be cooled or heated at room temperature, but preferably 0 to 100. ° C, most preferably 4-80 ° C. These polysaccharide solutions may be filtered through a centrifugal separator or a membrane filter if there are insolubles. For example, it may be processed with a pore size of 3,000 rpm for 10 minutes or more for centrifugation and 0.2 μm to 10 μm for a membrane filter.
次に、使用する多糖類は単独または複数の多糖類を混合して使用することもできる。混合に際して中性多糖同士の組み合わせはもちろん良好である。それ以外では、好ましくは中性多糖に対して塩基性多糖或いは酸性多糖の組み合わせであり、塩基性多糖と酸性多糖との組み合わせは凝集沈殿を起こすので避ける必要がある。また、多糖類単独での繊維作成も可能であるが、性能を最大限に発揮させるために必要に応じて、添加物として、合成高分子や繊維の改質剤、生理活性物質（細胞接着活性因子、細胞増殖因子、繊維芽細胞成長因子、免疫活性因子、神経作用因子等）、血清成分、生物組織成分、界面活性剤等を加える方が望ましい。例えば、多糖類以外の添加物としては、ポリエチレングリコール、ポリビニルアルコール、ポリビニルピロリドン、ポリ乳酸、絹フィブロイン、プロテオグリカン、フィブロネクチン、ヴィトロネクチン、エンタネクチン、エラスチン、ラミニン、セレクチン、ガレクチン、レクチン（WGA、コンカナバリンA等）、コラーゲン、ゼラチン、酵素（コラゲナーゼ、トリプシン、グルコシダーゼ、プロテインキナーゼ、ウロキナーゼ、SOD等）、アルブミン、フィブリン、フィブリノーゲン、ポリ‐L‐リジン、ポリ‐L- グルタミン酸、細胞外マトリクスタンパク質、インテグリン、アミノ酸（グリシン、プロリン、ヒドロキシプロリン、アラニン、セリン、アスパラギン、グルタミン酸、スレオニン、システイン、ロイシン、メチオニン等）、ジペプチド、トリペプチド、ペプチドタンパク質（チロシン‐イソロイシン‐グリシン‐セリン‐アルギニン配列、アルギニン‐グルタミン酸‐アスパラギン酸‐バリン配列、アルギンニン‐グリシン‐アスパラギン酸配列を含む）、グリコサミノグリカン、ムチン型結合糖鎖、アスパラギン型結合糖鎖、オリゴ糖（トレハロース、キチンオリゴ糖、キトサンオリゴ糖、ガラクトオリゴ糖、フルクトオリゴ糖、キシロオリゴ糖、シクロデキストリン等）、単糖（マンノース、N-アセチル-D-グルコサミン、グルコサミン、N-アセチル-D-ガラクトサミン、シアル酸、ムラミン酸、グルコース、ガラクトース、ラクトース、フコース、アラビノース等）、ガングリオシド、スフィンゴ脂質、糖脂質（アルキルグリコシド、ガラクトシルセラミド等）、ショ糖脂肪酸エステル、脂肪酸エステル（DHA、EPA等）、長鎖脂肪酸（アラキドン酸、リノール酸、レチノイン酸等）、グリセロール、多価アルコール、コレステロール、スクアレン、プロスタグランジン、トロンボキサン、ロイコトリエン、ステロイド、インシュリン、トランスフェリン、デキサメタゾン、ヒドロコルチゾン、チロキシン、トリヨードチロシン、β-メルカプトエタノール、アセチルコリン、酸類（乳酸、リンゴ酸、アスコルビン酸、グルコン酸、グルクロン酸、パントテン酸、ピルビン酸等）、グリチルリチン、ルチン、ステビオシド、サポニン、アルブチン、ポリフェノール（カテキン）、SOD様活性物質、インターフェロン、インターロイキン、サイトカイン、ケモカイン、TNF‐α、モノクローナル抗体、ポリクローナル抗体、補体因子、遺伝子、DNA、RNA、アデニン、牛胎仔血清、ビタミン類、無機塩類、シリコン、セラミック、アパタイト、ポリオキシエチレンソルビタンモノオレエート、エチレンジアミンテトラ酢酸等がある。添加物は溶解しても粉末で使用しても構わないが、好ましくは溶解する。添加物の溶媒としては、水、酸、アルカリ、エタノール、メタノール、アセトン、トルエン等があげられるが、添加物の溶解性を考慮して選択すればよく、少なくとも1種類、あるいは複数使用しても何ら問題にはならないが、好ましくは弱酸〜中性〜弱アルカリの溶媒であって、精製水やエタノール水、生理食塩水、各種緩衝液（リン酸緩衝液、酢酸緩衝液等）が望ましい。 Next, the polysaccharide to be used can be used singly or as a mixture of a plurality of polysaccharides. Of course, the combination of neutral polysaccharides is good during mixing. In other cases, the neutral polysaccharide is preferably a combination of a basic polysaccharide or an acidic polysaccharide with respect to the neutral polysaccharide, and the combination of the basic polysaccharide and the acidic polysaccharide causes aggregation and precipitation and should be avoided. In addition, it is possible to make fibers with polysaccharides alone, but as necessary, synthetic polymers and fiber modifiers, bioactive substances (cell adhesion activity) can be used to maximize performance. Factor, cell growth factor, fibroblast growth factor, immune activity factor, nerve action factor, etc.), serum component, biological tissue component, surfactant and the like are desirable. For example, additives other than polysaccharides include polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polylactic acid, silk fibroin, proteoglycan, fibronectin, vitronectin, enterectin, elastin, laminin, selectin, galectin, lectin (WGA, concanavalin). A), collagen, gelatin, enzyme (collagenase, trypsin, glucosidase, protein kinase, urokinase, SOD, etc.), albumin, fibrin, fibrinogen, poly-L-lysine, poly-L-glutamic acid, extracellular matrix protein, integrin, Amino acids (glycine, proline, hydroxyproline, alanine, serine, asparagine, glutamic acid, threonine, cysteine, leucine, methionine, etc.), dipipe Tide, tripeptide, peptide protein (including tyrosine-isoleucine-glycine-serine-arginine sequence, arginine-glutamic acid-aspartic acid-valine sequence, arginine-glycine-aspartic acid sequence), glycosaminoglycan, mucin-type glycan , Asparagine-type binding sugar chain, oligosaccharide (trehalose, chitin oligosaccharide, chitosan oligosaccharide, galactooligosaccharide, fructooligosaccharide, xylooligosaccharide, cyclodextrin, etc.), monosaccharide (mannose, N-acetyl-D-glucosamine, glucosamine, N -Acetyl-D-galactosamine, sialic acid, muramic acid, glucose, galactose, lactose, fucose, arabinose, etc.), ganglioside, sphingolipid, glycolipid (alkyl glycoside, galactosylceramide, etc.) Fatty acid ester, fatty acid ester (DHA, EPA, etc.), long chain fatty acid (arachidonic acid, linoleic acid, retinoic acid, etc.), glycerol, polyhydric alcohol, cholesterol, squalene, prostaglandin, thromboxane, leukotriene, steroid, insulin, Transferrin, dexamethasone, hydrocortisone, thyroxine, triiodotyrosine, β-mercaptoethanol, acetylcholine, acids (lactic acid, malic acid, ascorbic acid, gluconic acid, glucuronic acid, pantothenic acid, pyruvic acid, etc.), glycyrrhizin, rutin, stevioside, saponin , Arbutin, polyphenol (catechin), SOD-like active substance, interferon, interleukin, cytokine, chemokine, TNF-α, monoclonal antibody, polyclonal antibody, Body factor, gene, DNA, RNA, adenine, bovine calf serum, vitamins, inorganic salts, silicon, ceramics, apatite, polyoxyethylene sorbitan monooleate, is ethylenediaminetetraacetic acid or the like. The additive may be dissolved or used as a powder, but is preferably dissolved. Examples of the solvent for the additive include water, acid, alkali, ethanol, methanol, acetone, toluene, and the like, which may be selected in consideration of the solubility of the additive, and at least one kind or a plurality may be used. Although it does not cause any problem, it is preferably a weak acid to neutral to weak alkali solvent, and purified water, ethanol water, physiological saline, and various buffers (phosphate buffer, acetate buffer, etc.) are desirable.
キャピラリーの口径は、目的に応じて自由に選択すればよく、特に限定するものではないが実施例では50μmの口径を使用している。基板は導電性のある素材であればよく、アルミニウム等がよく使われるが、その他の導電性素材であっても構わない。キャピラリーから基板までの距離は、自由に選択すればよく、特に限定するものではないが、実施例では30mm〜100mmとしている。印加電圧は、溶液の性状によって変えればよく、電圧が高いほど細長い繊維が得やすく、低い場合には繊維構造になりにくく、短い繊維や粒子が得やすく、好ましくは2,000v〜50,000vであるが、最も好ましくは4,000v〜20,000vである。溶液の粘性が高い場合には、印加電圧をあげれば良いが、シリンジポンプ等で補助的に上部から加圧してもよく、得られる繊維や被膜の性質等の大きな影響はなく何ら問題にはならない。その他に繊維や薄膜に影響するものとしては、装置内の湿度があるが、湿度70％以上になると安定的に紡糸することができないので適用は70％未満であり、好ましくは10％〜30％で一定に保つ必要がある。 The diameter of the capillary may be freely selected according to the purpose, and is not particularly limited. In the embodiment, a diameter of 50 μm is used. The substrate may be any conductive material, and aluminum or the like is often used, but other conductive materials may be used. The distance from the capillary to the substrate may be freely selected and is not particularly limited, but is set to 30 mm to 100 mm in the embodiment. The applied voltage may be changed depending on the properties of the solution. The higher the voltage, the easier to obtain elongated fibers, and when it is low, the fiber structure is less likely to form, making it easier to obtain short fibers and particles, preferably 2,000 v to 50,000 v. Most preferably, it is 4,000v to 20,000v. If the viscosity of the solution is high, the applied voltage may be increased, but it may be supplementarily pressurized from above with a syringe pump, etc., and there is no significant effect on the properties of the resulting fiber or coating, and there is no problem. . Other factors that affect the fiber and thin film include the humidity inside the device, but if the humidity exceeds 70%, it cannot be stably spun, so the application is less than 70%, preferably 10% to 30%. It is necessary to keep it constant.
基板に集積された繊維や不織性の薄膜は、強度を得るために架橋しても構わない。架橋剤としてはグルタルアルデヒド、カルボジイミド、カルボニルイミダゾール、ジエポキシ化合物、ジカルボン酸無水物、エピクロルヒドリン等がある。これら架橋した後は、アセトン、エタノール、精製水や生理食塩水、各種緩衝液等で架橋剤が残存しないように洗浄する。 The fibers and non-woven thin film integrated on the substrate may be cross-linked to obtain strength. Examples of the crosslinking agent include glutaraldehyde, carbodiimide, carbonyl imidazole, diepoxy compound, dicarboxylic acid anhydride, epichlorohydrin and the like. After these cross-linkings, washing is performed with acetone, ethanol, purified water, physiological saline, various buffer solutions or the like so that the cross-linking agent does not remain.
本装置の原理を応用すれば、どんな形状や大きさの基板でも噴射でき、ナノスケールの繊維を基材上へ堆積させることができる。また、基板に直接噴霧するのではなく、基材としてガラス、プラスチック、不織布等に直接噴霧し表面にコーティングすることもできる。例えば、細胞培地の基材や生体材料（人工弁、人工臓器、人工血管、人工骨、人工歯、人工皮膚等）の材料の表面に直接噴霧することもできる。また、異なる溶液を調製し、同一基板上にそれぞれ噴霧処理することで性質の異なる薄膜を幾重にも重ねることもできる。例えば、合成高分子層（支持層）と多糖類層からなる2層構造の薄膜も可能である。また薄膜を基板から剥がし、被覆材として創傷患部である生体組織に貼り付けたり、埋没させたりもできる。 By applying the principle of this apparatus, substrates of any shape and size can be jetted and nanoscale fibers can be deposited on a substrate. Further, instead of spraying directly on the substrate, it is possible to directly spray glass, plastic, nonwoven fabric or the like as a base material to coat the surface. For example, it can be directly sprayed on the surface of a cell culture medium material or a biomaterial (artificial valve, artificial organ, artificial blood vessel, artificial bone, artificial tooth, artificial skin, etc.). Also, different solutions can be prepared and sprayed on the same substrate, so that thin films with different properties can be stacked several times. For example, a thin film having a two-layer structure including a synthetic polymer layer (support layer) and a polysaccharide layer is also possible. Further, the thin film can be peeled off from the substrate, and can be affixed to or embedded in a living tissue that is a wounded part as a covering material.
多糖類のナノスケールの繊維およびその繊維からなる不織性の薄膜は、従来の多糖繊維よりも極細なので比表面積が大きく、細胞への接着面積が広がる。細胞の増殖や分化に最適な足場となり、再生医療分野における医用材料である生体組織培養の基材および人工臓器や創傷被覆材等の生体材料の一部として利用できる。 Polysaccharide nanoscale fibers and non-woven thin films made of the fibers are finer than conventional polysaccharide fibers, and therefore have a large specific surface area and an increased adhesion area to cells. It becomes an optimal scaffold for cell proliferation and differentiation, and can be used as a base material for biological tissue culture, which is a medical material in the field of regenerative medicine, and as a part of biological materials such as artificial organs and wound dressings.
以下に本発明の実施例を記載するが、本発明はこれらに何ら限定されるものではない。 Examples of the present invention will be described below, but the present invention is not limited thereto.
図１記載の静電紡糸装置を用いた。試料はカニ殻から分離精製されたキトサン（Mw12万、株式会社共和テクノス製）を用いた。試料の濃度は10％である（w/v，溶解剤として酢酸を5%含む）。この試料駅（粘度120cp/20℃）を先端口径が50μmのキャピラリーに充填し、電圧を印加した（室温下、大気圧、湿度20％）。その結果、アルミニウム基板(1cm×1cm)上にナノスケールの繊維からなる不織性の薄膜が観察された(図２)。 The electrospinning apparatus shown in FIG. 1 was used. The sample used was chitosan (Mw 120,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shells. The sample concentration is 10% (w / v, containing 5% acetic acid as solubilizer). The sample station (viscosity 120 cp / 20 ° C.) was filled into a capillary with a tip diameter of 50 μm, and voltage was applied (at room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 2).
図１記載の静電紡糸装置を用いた。試料はカニ殻から分離精製されたキトサン（Mw7万、株式会社共和テクノス製）を用いた。試料液の濃度は10％である（w/v，溶解剤として酢酸を5％含む）。この試料液に添加剤として5％ポリエチレングリコール（Mw50万、和光純薬工業株式会社製）を6：4の重量比で加え（粘度120cp/20℃）、先端口径が50μmのキャピラリーに充填し、印加電圧は9.13kVで実施した（室温下、大気圧、湿度20％）。その結果、アルミニウム基板（1cm×1cm）上に直径98-386nmのナノスケールの繊維からなる不織性の薄膜が観察された（図３）。 The electrospinning apparatus shown in FIG. 1 was used. The sample used was chitosan (Mw 70,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shell. The concentration of the sample solution is 10% (w / v, containing 5% acetic acid as a solubilizer). 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added to this sample solution at a weight ratio of 6: 4 (viscosity 120 cp / 20 ° C), and the capillary was filled with a tip diameter of 50 μm. The applied voltage was 9.13 kV (room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers with a diameter of 98-386 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 3).
図１記載の静電紡糸装置を用いた。試料はサメ軟骨から分離精製されたコンドロイチン硫酸ナトリウム（Mw11万、株式会社堺商事製）を用いた。試料液の濃度は10％である（w/v，蒸留水で溶解）。この試料液に添加剤として5％ポリエチレングリコール（Mw50万、和光純薬工業株式会社製）を8：2の重量比で加え（粘度78cp/70℃）、先端口径が50μmのキャピラリーに充填し、印加電圧は16.53kVで実施した（室温下、大気圧、湿度20％）。その結果、アルミニウム基板（1cm×1cm）上に直径115-304nmのナノスケールの繊維からなる不織性の薄膜が観察された（図４）。 The electrospinning apparatus shown in FIG. 1 was used. The sample used was sodium chondroitin sulfate (Mw 110,000, manufactured by Sakai Shoji Co., Ltd.) separated and purified from shark cartilage. The concentration of the sample solution is 10% (w / v, dissolved in distilled water). To this sample solution, 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added at a weight ratio of 8: 2 (viscosity 78 cp / 70 ° C.) and filled into a capillary with a tip diameter of 50 μm. The applied voltage was 16.53 kV (room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers having a diameter of 115 to 304 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 4).
図１記載の静電紡糸装置を用いた。試料は柑橘類から分離精製されたペクチン（Mw51万、和光純薬工業株式会社製）を用いた。試料液の濃度は5％である（w/v，蒸留水で溶解）。この試料液に添加剤として5％ポリエチレングリコール（Mw50万、和光純薬工業株式会社製）を6：4の重量比で加え（粘度26cp/70℃）、先端口径が50μmのキャピラリーに充填し、印加電圧は10.89kVで実施した（室温下、大気圧、湿度20％）。その結果、アルミニウム基板（1cm×1cm）上に直径75-267nmのナノスケールの繊維からなる不織性の薄膜が観察された（図５）。 The electrospinning apparatus shown in FIG. 1 was used. As a sample, pectin separated from citrus fruits (Mw 510,000, manufactured by Wako Pure Chemical Industries, Ltd.) was used. The concentration of the sample solution is 5% (w / v, dissolved in distilled water). To this sample solution, 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added at a weight ratio of 6: 4 (viscosity 26 cp / 70 ° C.), and filled into a capillary with a tip diameter of 50 μm. The applied voltage was 10.89 kV (at room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers having a diameter of 75 to 267 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 5).
図１記載の静電紡糸装置を用いた。試料はカニ殻から分離精製されたキトサン（Mw3万、株式会社共和テクノス製）を用いた。試料液の濃度は10％である（w/v，溶解剤として酢酸を5％含む）。この試料液に添加剤として15％ポリビニルピロリドン（Mw36万、和光純薬工業株式会社製）を1：1の重量比で加え（粘度120cp/20℃）、先端口径が50μmのキャピラリーに充填し、印加電圧は14.00kVで実施した（室温下、大気圧、湿度20％）。その結果、アルミニウム基板（1cm×1cm）上に直径70‐210nmのナノスケールの繊維からなる不織性の薄膜が観察された（図６）。 The electrospinning apparatus shown in FIG. 1 was used. As a sample, chitosan (Mw 30,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shells was used. The concentration of the sample solution is 10% (w / v, containing 5% acetic acid as a solubilizer). 15% polyvinylpyrrolidone (Mw 360,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added to this sample solution at a weight ratio of 1: 1 (viscosity 120 cp / 20 ° C), and the capillary was filled with a tip diameter of 50 μm. The applied voltage was 14.00 kV (at room temperature, atmospheric pressure, humidity 20%). As a result, a non-woven thin film composed of nanoscale fibers having a diameter of 70-210 nm was observed on an aluminum substrate (1 cm × 1 cm) (FIG. 6).
（自立薄膜の作製）
図１記載の静電紡糸装置を用いた。試料はカニ殻から分離精製されたキトサン（Mw3万、株式会社共和テクノス製）を用いた。試料液の濃度は10％である（w/v，溶解剤として酢酸を5％含む）。この試料液に添加剤として5％ポリエチレングリコール（Mw50万、和光純薬工業株式会社製）を8：2の重量比で加え（粘度115cp/22℃）、先端口径が50μmのキャピラリーに充填し、基板上にアルミ箔を敷き、印加電圧は16kVで実施した（室温下、大気圧）。装置内の湿度が異なる条件下で実施した結果、繊維径の異なる2種類の薄膜を得た。それぞれの薄膜について繊維径を測定したところ直径100-300nmの薄膜（湿度20%）と直径500-1000nmの薄膜（湿度35%）であった。次ぎに、薄膜はアルミ箔に付着状態のままメタノールと１N苛性ソーダ（重量比99：1）の混合溶媒に室温で2時間浸漬した後、アルミ箔から剥がした。更に純水で洗浄し室温で風乾して不溶性の自立膜を得た。
(Production of free-standing thin film)
The electrospinning apparatus shown in FIG. 1 was used. As a sample, chitosan (Mw 30,000, manufactured by Kyowa Technos Co., Ltd.) separated and purified from crab shells was used. The concentration of the sample solution is 10% (w / v, containing 5% acetic acid as a solubilizer). To this sample solution, 5% polyethylene glycol (Mw 500,000, manufactured by Wako Pure Chemical Industries, Ltd.) as an additive was added at a weight ratio of 8: 2 (viscosity 115 cp / 22 ° C.), and filled in a capillary with a tip diameter of 50 μm. An aluminum foil was laid on the substrate, and the applied voltage was 16 kV (room temperature, atmospheric pressure). As a result of carrying out under conditions where the humidity in the apparatus was different, two types of thin films with different fiber diameters were obtained. When the fiber diameter was measured for each thin film, it was a thin film with a diameter of 100-300 nm (humidity 20%) and a thin film with a diameter of 500-1000 nm (humidity 35%). Next, the thin film was immersed in a mixed solvent of methanol and 1N caustic soda (weight ratio 99: 1) at room temperature for 2 hours while being attached to the aluminum foil, and then peeled off from the aluminum foil. Further, it was washed with pure water and air-dried at room temperature to obtain an insoluble self-supporting film.
(タンパク質吸着試験)
本試験にはタンパク質として牛血清アルブミン（シグマ製）を用い、蒸留水で1%牛血清アルブミン溶液とした。その溶液を乾燥した薄膜（湿度20%条件、繊維径100nm- 300nm）に対して37倍量(重量)添加し浸漬した。浸漬液は蒸留水で10倍量に希釈後、遊離のタンパク質を280nmによる吸光度法で測定し、牛血清アルブミン検量線から薄膜に吸着したタンパク量を求めた。また、乾燥した薄膜（湿度35%条件、繊維径500nm- 1000nm）についても1%牛血清アルブミン溶液を50倍量(重量)添加し浸漬し、10倍に希釈後、同様に、遊離のタンパク質を280nmによる吸光度法で測定し、牛血清アルブミン検量線から薄膜に吸着したタンパク量を求めた。
その結果、繊維径100nm- 300nmの薄膜は乾燥薄膜1g当たり452mgの牛血清アルブミンを吸着し、繊維径500nm- 1000nmの薄膜は乾燥薄膜1g当たり171mgの牛血清アルブミンを吸着した。繊維径の細い薄膜は繊維径の太い薄膜よりも約2.6倍量の吸着量が高かった。
(Protein adsorption test)
In this test, bovine serum albumin (manufactured by Sigma) was used as a protein, and a 1% bovine serum albumin solution was prepared with distilled water. The solution was immersed in a 37-fold amount (weight) of the dried thin film (humidity 20% condition, fiber diameter 100 nm-300 nm). The soaking solution was diluted 10 times with distilled water, free protein was measured by an absorbance method using 280 nm, and the amount of protein adsorbed on the thin film was determined from a calibration curve of bovine serum albumin. Also, add 50% (weight) of 1% bovine serum albumin solution to the dried thin film (humidity 35% condition, fiber diameter 500nm-1000nm), soak it and dilute it 10 times. The amount of protein adsorbed on the thin film was determined from the bovine serum albumin calibration curve by measuring the absorbance at 280 nm.
As a result, a thin film with a fiber diameter of 100 nm to 300 nm adsorbed 452 mg of bovine serum albumin per 1 g of the dry thin film, and a thin film with a fiber diameter of 500 nm to 1000 nm adsorbed 171 mg of bovine serum albumin per 1 g of the dry thin film. The thin film with a small fiber diameter was about 2.6 times more adsorbed than the thin film with a large fiber diameter.
［比較例］
比較例として、一般に流通しているキトサン繊維（不織布）を入手したもので、繊維の直径は12‐38μmであった（図７）。
[Comparative example]
As a comparative example, a commercially available chitosan fiber (nonwoven fabric) was obtained, and the fiber diameter was 12-38 μm (FIG. 7).
本発明の静電紡糸法による多糖類のナノスケールの繊維およびその繊維によって構成された成形体は、安全性が高く、細胞毒性がなく、しかも比表面積が大きくなるので、医療分野、特に再生医療分野における医用材料として、生体組織培養の基材や生体材料（人工臓器、人工血管、人工骨等）の足場、および創傷被覆材等に利用できる。 The nano-scale fibers of polysaccharides by the electrospinning method of the present invention and the molded body constituted by the fibers are highly safe, non-cytotoxic, and have a large specific surface area. As a medical material in the field, it can be used as a base material for biological tissue culture, a scaffold for biological materials (artificial organs, artificial blood vessels, artificial bones, etc.), a wound dressing, and the like.
１ 電圧（加電圧を制御）
２ 電極
３ キャピラリー
４ 基板
５ アース
1 Voltage (Controlling applied voltage)
2 Electrode 3 Capillary 4 Substrate 5 Ground
Claims (7)
A medical material comprising the nonwoven film of claim 4 or 5.
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